Methods and apparatus for fractionation-based chemical analyses

ABSTRACT

A method for analyzing chemicals includes fractionating a complex sample into at least two sample portions that each include portions of two polypeptides though in different concentration ratios, digesting and performing LC/MS on each of the sample portions, and associating precursor ions observed via LC/MS with their corresponding polypeptide in response to LC/MS-provided intensity data. A set of precursor-ions that has substantially similar intensity ratios in both sample portions is determined to be associated with the same polypeptide.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/916,320 filed Jun. 10, 2009, now U.S. Pat. No. 8,143,066, which isthe National Stage of International Application No. PCT/US2006/21771,filed Jun. 5, 2006, which is hereby incorporated by reference.

TECHNICAL FIELD

The invention generally relates to analyses of compounds, and, moreparticularly, to instruments and methods for polypeptide analyses.

BACKGROUND INFORMATION

Proteomics generally refers to studies involving complex mixtures ofproteins derived from biological systems. Proteomic studies often focuson identification of proteins or determination of changes in the stateof a biological system. Identification and quantification of proteins incomplex biological samples is a fundamental problem in proteomics.

Liquid chromatography coupled with mass spectrometry (LC/MS) has becomea fundamental tool in proteomic studies. Separation of intact proteinsor of their proteolyzed peptide products by liquid chromatography (LC)and subsequent analysis by mass spectrometry (MS) forms the basis ofmany common proteomic methodologies. Methods that measure changes in theexpression level of proteins are of great interest as they can form thebasis of biomarker discovery and clinical diagnostics.

Rather than directly analyzing intact proteins, proteins of aretypically digested to produce a specific set of proteolytic peptides.The resulting peptides are then often characterized via LC/MS analysis.A common enzyme used for digestion is trypsin. In tryptic digestion, theproteins present in a complex mixture are cleaved to produce peptides asdetermined by the cleavage specificity of the proteolytic enzyme. Fromthe identity and concentration of the observed peptides, availablealgorithms serve to identify and quantify the proteins in the sample.

In LC/MS analysis, the peptide digest is first separated and analyzed byLC separation followed by MS analysis. Ideally, the mass of a singlepeptide, measured with sufficient accuracy, provides a uniqueidentification of the peptide. In practice, however, achieved massaccuracies typically are on the order of 10 ppm or larger. In general,such mass accuracy is not sufficient to uniquely identify a peptideusing the mass measurement alone.

For example, in the case of a mass accuracy of 10 ppm, on the order of10 peptide sequences are identified in a search of a typical database ofpeptides sequences. This number of sequences would increasesignificantly if search restraints on mass accuracy were lowered, orsearches for chemical or post-translational modifications, losses of H₂Oor NH₃, and point mutations were allowed, for example. Thus, if apeptide's sequence is modified by either a deletion or substitution, useof only the precursor's mass for identification of the petite will leadto a false identification. A further complication arises from thepossibility that two peptides can have the same amino acid compositionbut have different sequences.

In the case of peptide precursors, product fragments can be obtained byfragmentation at a single peptide bond in the precursor. Such a singlefragmentation produces two sub-sequences. The fragment containing thepeptide's C-terminal, if ionized, is termed a Y-ion, and the fragmentcontaining the peptide's N-terminal, if ionized is termed a B-ion.

Proteins are often identified by comparing analysis data to a databasethat associates protein identities with information about fragments ofthe proteins, such as masses of the fragments. For example, if atheoretical peptide mass from a database lies within a mass searchwindow of the mass of a precursor measured in the data, it is deemed ahit.

The search can provide a list of possible matching peptides found in thedatabase. These possible matching database peptides may or may not beweighted by statistical factors. The possible outcomes of such a searchare that no possible matching database peptides are identified, onepossible matching database peptide is identified, or more than onepossible matching database peptide are identified. The higher theresolution of the MS, assuming proper instrument calibration, thesmaller the ppm threshold, and consequently, the fewer the falseidentifications. If there are one or more matches to the peptides in thedatabase, peptide-fragment ion data may be used to validate a match.

During a search, multiple charge states and multiple isotopes can besearched. Further, empirically produced confidence rules can be appliedto help identify valid matches.

SUMMARY OF THE INVENTION

Some embodiments of the invention arise from the realization thatoverlapping polypeptide-related chromatographic peaks can bedeconvoluted through oversampling fractionation of the overlappingpeaks. In particular, comparisons of MS data derived from oversampledmaterials often provides a more complete analysis than available withoutoversampling.

Accordingly, an example embodiment features a method for chemicalanalyses. The method includes fractionating a complex sample into atleast a first sample portion and a second sample portion that eachinclude portions of two polypeptides though in different concentrationratios, digesting and performing LC/MS on each of the sample portions,and associating precursor ions observed via LC/MS with theircorresponding one of the polypeptides in response to LC/MS-provideddata. The precursor ions are associated with their correspondingpolypeptides by comparison of, for example, precursor-ion intensities.For example, a set of precursor-ions that has substantially similarintensity ratios in both sample portions is determined to be associatedwith the same polypeptide.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating the principles of the invention.

FIG. 1 is a flow diagram of a method for performing chemical analyses ofcompounds, in accordance with one embodiment of the invention;

FIG. 2 is a partial flow diagram of a method that is related to themethod of FIG. 1;

FIG. 3 is a flow diagram of a method for chemical analyses, inaccordance with one embodiment of the invention;

FIG. 4 is a three-dimensional graphical diagram of an example profileobtained from a sample transalodase, in accordance with one embodimentof the invention;

FIG. 5 is a two-dimensional bar graph of the profile of FIG. 4, showingintensity of precursor ions and product ions as a function of ion mass;

FIG. 6 is a two-dimensional bar graph of the profile of FIG. 4, showingintensity of precursor ions and product ions as a function ofretention-time;

FIG. 7 is a two-dimensional plot corresponding to a top-down view of theprofile of FIG. 4;

FIG. 8 is a flow diagram of a method for chemical analyses, inaccordance with one embodiment of the invention;

FIG. 9A is a block diagram of an LC/MS system, in accordance with oneembodiment of the invention;

FIG. 9B shows three related graphs, which illustrate the collection ofmass spectra in accordance with one embodiment of the invention;

FIG. 10 and FIG. 11 are flow diagrams of processing steps that involveretention time matching of a precursor and its related product ions, inaccordance with one embodiment of the invention;

FIG. 12 is a graphical representation of a reference injection, inaccordance with one embodiment of the invention;

FIG. 13A is a graphical representation of a first target injection, inaccordance with one embodiment of the invention;

FIG. 13B is graphical representation of an alignment or normalization ofthe first target injection of FIG. 13A and the reference injection ofFIG. 12 in accord with a common precursor;

FIG. 14A, shown is a graphical representation of a second targetinjection;

FIG. 14B is graphical representation of an alignment or normalization ofthe second target injection of FIG. 14A and the reference injection ofFIG. 12 in accord with the common precursor;

FIG. 15 is a graphical representation of an output spectrum including aprecursor and associated product ions, in accordance with one embodimentof the invention;

FIG. 16 is a graphical representation of a reference injection, inaccordance with one embodiment of the invention; and

FIG. 17 is a graphical representation of a first target injection foranalysis with the reference injection of FIG. 16.

DESCRIPTION

As used herein, the following terms generally refer to the indicatedmeanings:

Protein—a specific primary sequence of amino acids assembled as a singlepolypeptide.

Peptide—a specific sequence of amino acids assembled as a singlepolypeptide contained within the primary sequence of a protein.

Tryptic peptides—peptides generated from a protein sequence that resultfrom enzymatic cleavage of the protein by trypsin. In the ensuingdescription, digest peptides are referred to as tryptic peptides forconvenience. It should be understood, however, that embodiments of thepresent invention apply to other peptide digestion techniques. Moreover,the term “digestion” is used herein to refer generally to any suitablemethod for degrading or cleaving a polypeptide, including, for example,the use of cellular enzymes (proteases) and intramolecular digestion.The term “proteolytic,” as used herein, refers to any enzyme whichdigests or lyses large proteins into smaller sections or amino acids.

Precursor peptides —tryptic peptides (or other protein cleavageproducts) that are generated using a protein-cleavage protocol. Theprecursors are optionally separated chromatographically and passed to amass spectrometer. An ion source ionizes these precursor peptides totypically produce a positively charged, protenated form of theprecursor. The mass of such positively charged protenated precursor ionis herein referred as the “mwHPlus” or “MH+” of the precursor. In thefollowing, the term “precursor mass” refers generally to the protenated,mwHPlus or MH+ mass of the ionized, peptide precursor.

Fragments—Multiple types of fragments can occur in LC/MS analyses. Inthe case of tryptic peptide precursors, fragments can include polypetideions that are produced from collisional fragmentation of the intactpeptide precursors and whose primary amino acid sequence is containedwithin the originating precursor peptide. Y-ions and B-ions are examplesof such peptide fragments. Fragments of tryptic peptides can alsoinclude immonium ions, functional groups such as a phosphate ion (PO₃),mass tags cleaved from a specific molecule or class of molecules, or“neutral loss” of water (H₂O) or ammonia (NH₃) molecules from theprecursor.

Y-ions and B-ions—If a peptide fragments at the peptide bond, and if acharge is retained on the N terminal fragment, that fragment ion istermed a B-ion. If the charge is retained on the C terminal fragment,the fragment ion is termed a Y-ion. A more comprehensive list ofpossible fragments and their nomenclature is provided in Roepstorff andFohlman, Biomed Mass Spectrom, 1984; 11(11):601 and Johnson et al, Anal.Chem. 1987, 59(21): 2621:2625.

Retention time—in context, typically refers to the point in achromatographic profile at which an entity reaches its maximumintensity.

Ions—each peptide typically appears in an LC/MS analysis as an ensembleof ions due to the natural abundance of the isotopes of the constituentelements. An ion has a retention time and an m/z value. The massspectrometer (MS) detects only ions. The LC/MS technique produces avariety of observed measurements for every detected ion. This includes:the mass-to-charge ratio (m/z), mass (m), the retention time, and thesignal intensity of the ion, such as a number of ions counted.

MwHPlus—The neutral, monoisotopic mass of the peptide plus the weight ofone proton, 1.007825 amu.

Generally, an LC/MS analysis optionally provides an empiricaldescription of a peptide in terms of its mass, charge, retention timeand total intensity. When a peptide elutes from the chromatographiccolumn, it elutes over a specific retention time period and reaches itsmaximum signal at a single retention time. After ionization and(possible) fragmentation, the peptide appears as a related set of ions.The different ions in the set correspond to different isotopiccompositions and charges of the common peptide. Each ion within therelated set of ions produces a single peak retention time and peakshape. Since these ions originate from a common peptide, the peakretention time and peak shape of each ion is identical, within somemeasurement tolerance. The MS acquisition of each peptide producesmultiple ion detections for all isotopes and charge states, all sharingthe same peak retention-time and peak shape within some measurementtolerance.

In an LC/MS separation, a single peptide (precursor or fragment)produces many ion detections, which appears as a cluster of ions, atmultiple charge states. Deconvolution of these ion detections from sucha cluster, indicates the presence of a single entity of a uniquemonoisotopic mass, at a specific retention time, of a measured signalintensity, in a charge state.

Protein Database—In some embodiments of the present invention, ananalyst utilizes a database of proteins. In a typical database, eachincluded protein is described by its primary sequence of amino acids. Ananalyst might choose a database that is intended to closely matchproteins under study. For example, an E. Coli database could be comparedto data obtained from a cell lycate of E. Coli. Similarly, a human serumdatabase could be compared to data obtained from human serum. A usercould choose a subset database. A user could choose a superset database,such as all proteins listed in the SwissProt database produced by theSwiss-Prot groups at the Swiss Institute of Bioinformatics (SIB) and theEuropean Bioinformatics Institute (EBI). A user could choose a data abase that contains simulated proteins, described by random sequences ofamino acids. Such random databases are used in control studies toevaluate or calibrate protein identification systems and searchalgorithms. A user could choose a database that combines both naturallyoccurring and artificial sequences. From the protein database, one caninfer from each sequence, the sequence and masses of tryptic precursorions, Y- and B-ions, and other possible fragment ions that would resultfrom those precursors.

FIG. 1 is a flow diagram of a method 100 for performing chemicalanalyses of compounds. The method 100 includes digesting 110 one or morecompounds of a reference sample into component fragments of thecompounds, separating 120 the components, ionizing 130 and massanalyzing 140 at least some of the separated components, and storing 150in a catalog of profiles a profile of at least one compound in thesample.

The method optionally includes repeating 160 on a subject sample thesteps of digesting 110, separating 120, ionizing 130 and mass analyzing140, and identifying 170 the one or more compounds in the subject samplein response to a match between subject-sample analysis data and datastored in the profiles of the one or more compounds.

Preferably, for repeating 160 analysis steps on the subject sample,effectively the same pre-selected method(s) for digestion,chromatographic separation, and/or ionization are used for digesting110, separating 120, ionizing 130, and mass analyzing 140 of the subjectsample as used for the reference sample. Through use of substantiallythe same methods, subject-sample data is more accurately compared to acatalog of profiles to identify 170 compounds in the subject sample.Thus, an analyst optionally develops her own catalog using her ownanalytical instruments and analysis methods or recipes, and thusproduces more reliably and easily obtains identifications than otherwisepossible through use of generic databases.

Optionally, through use of substantially the same methods, catalogsprepared by different analysts in different laboratories are combinedinto master catalogs. These catalogs are then useable by other analyststo identify proteins obtained through substantially the same methods.Thus, for example, different labs or different instruments in the samelab, using substantially the same equipment and methods, maycollaboratively produce catalogs of protein profiles. Such labs may alsobe users of such catalogs.

Some preferred uses of the method 100 are directed towardprotein-related analyses. Thus, for convenience, the followingdescription refers to proteins and related fragments, and utilizesexamples of analyses of compounds that are polypeptides, such asproteins; in these examples, a protein is digested into componentfragments that are precursor fragments of the protein. Precursors, inturn, are ionized to form precursor ions and optionally are themselvesfragmented into product ions in preparation for mass analysis.

Although the description focuses on examples related to polypeptides,such examples are not intended to limit the scope of the invention toanalyses of polypeptides; one having ordinary skill in thechemical-analysis arts will recognize that principles of the inventionare applicable to analyses of other chemical compounds.

Digesting 110 is accomplished via any suitable technique for cleavingproteins, including known techniques. For example, as described above, aprotein is digested into precursor polypeptides or amino acids throughuse of one or more enzymes such as trypsin. Fragments of a protein orpolypeptide are herein generally referred to as “precursors.” Such afragment is a precursor in the sense that it is optionally used inadditional analyses subsequent to chromatographic separation. Asdescribed in more detail below, precursor fragments are optionallyionized and/or further fragmented into product fragments.

Separating 120 is accomplished by any suitable chromatographic-relatedtechnique, including known techniques such as reverse-phasechromatography, gel-permeation chromatography, size-exclusionchromatography, and electrophoresis. Separating 120 provides valuesassociated with retention times of the proteins and/or precursorsobtained from digesting 110 proteins in a sample.

In preparation for mass analyzing 140 the eluent of a chromatographicseparation 120, the eluent from the separating 120 process is subjectedto an ionizing 130 process. Any suitable ionizing 130 process isoptionally used, including known techniques such as electrosprayionization and MALDI. During the ionizing 130 process, at least some ofthe precursors are ionized to form precursor ions. For example, a singleprotein molecule is digested 110 to form twenty precursor fragments, ofwhich ten are ionized during ionizing 130. Optionally, as described inmore detail below, precursors are further fragmented to obtain productions to assist in the identification of associated precursors.

Mass analyzing 140 provides values associated with mass and valuesassociated with ion intensity of the precursor ions. Mass analyzing 140is performed via any suitable mass-analysis techniques, including knowntechniques. Such techniques include magnetic-sector spectrometry andtime-of-flight spectrometry.

Information obtained from the above-described analysis is used to defineone or more profiles for one or more associated proteins in the sample.A protein profile is defined by values associated with retention time,ion mass, and ion intensity of precursor ions associated with theprotein. Optionally, the profile of the protein is also defined by theidentity of the protein. Some preferred embodiments include product-iondata in profiles. Thus, the profile of the protein may also be definedby values associated with retention time, ion mass, and ion intensity ofproduct ions associated with the precursors of the protein.

The profile is stored 150 in a catalog of profiles for later use indetecting, identifying and/or quantifying the protein in later analyzedsubject samples. Optionally, the profiles are defined in an existingprotein database by annotating the proteins listed in the database withvalues associated with retention time, ion mass, and ion intensity ofprecursor ions associated with the corresponding listed proteins.

Any suitable measure of ion-intensity value is used in the definition ofprofiles. For example a total number of ion counts in an LC/MS peak issuitably used, as will be understood by one having ordinary skill in thechemical-analysis arts.

The particular types of values used to define a protein profileoptionally are varied, for example, to suit a particular need of ananalyst. A profile is optionally defined, for example, with values ofm/z and/or precursor charge state and/or other values as will berecognized by one having ordinary skill.

The analysis and profile definition process are optionally repeated, asdesired. Thus, a catalog of protein profiles is developed that providesa convenient reference source for analysis of subject samples thatinclude known and/or unknown proteins. The catalog optionally includesmore than one profile for each of one or more proteins. Moreover, asdescribed in more detail below, subject samples themselves optionallyprovide data for additional protein profiles. Examples of some proteinprofiles are described below with reference to FIG. 4 through FIG. 7.

Once a catalog includes a profile for a particular protein, that proteinis available for detection, identification 170, and/or quantitation in asubject sample analyzed optionally by the same or sufficiently the sameanalysis steps described above. This approach provides sufficientlycomparable analysis data for the protein in the subject sample to beaccurately matched to the corresponding data in the profile in thecatalog. A confirmed match with the precursor-related data in theprofile confirms the detection of the protein in the subject sample.Optionally, a confirmed match with the product-related data in theprofile confirms the detection of the protein in the subject sample.Moreover, the intensity data obtained from the subject sample, incomparison to the intensity data in the profile, from precursors and/orproducts optionally provides quantification of the protein in thesubject sample.

A catalog is searched for possible matches in any suitable manner. Forexample, comparisons are iteratively made with each profile in a cataloguntil a best match, if any, is obtained. If a same LC method is used togenerate a catalog and to analyze a subject sample, retention-timevalues of subject-sample precursor ions are directly comparable toprofile values, using, for example, a pre-selected retention-timewindow. Product-ion retention times are optionally included in thiscomparison.

If different LC methods are used, such that retention-time values areless useful, comparison to mass-related values may still be used toassociate subject-sample precursor-ion data with corresponding proteinprofiles.

Generally, the intensity values of precursors in a subject sample willbe different than the values stored with a corresponding profile becauseof protein concentration differences. A scaling factor is optionallyapplied to provide effectively exact matches between subject-sampleprecursor ions and profile precursor ions. A determined scaling factoris optionally used for quantification of the protein in the subjectsample.

Generally, if a protein has a higher concentration in a subject samplethan it had in a reference sample that was used to define its profile,all precursors included in the profile will be observable in the subjectsample. Conversely, if the subject sample concentration is lowerrelative to the profile, some profile precursor ions may beundetectable.

After a correspondence between a subject sample's precursor-ion data anda profile is obtained, the match is optionally confirmed thoughadditional comparison steps. For example, an initial match based oncomparisons of data for three precursors is optionally verified bycomparing data for additional precursors and/or product ions. Forexample, a scale factor determined from the initial comparisons isusable to search for matches between values for the additionalcomponents of the profile. Thus, generally, additional available datashould be consistent with a preliminary match to validate thepreliminary match.

In some embodiments, data of a single precursor is used to obtain amatch to a profile, provided that the match includes a match betweenretention-time values. Without retention-time matching, data of at leasttwo precursor ions is preferably used. Often, good accuracy in matchingis obtained through use of data of three precursor ions. With aconfirmed match, the identity and concentration of the match protein areoptionally indicated. Preferably, precursor ions having greatestintensity values are used to define profiles and for comparisons tothose profiles. If product-ion data are included in the protein profile,and are observed in the sample, these ions can be matched to furtherconfirm the identify of the protein in the sample.

The reference sample optionally includes unknown and/or known proteins,and the method 100 optionally assists accurate identification ofproteins in the reference sample in support of a definition of a profilefor that protein. For example, FIG. 2 is a partial flow diagram of amethod 200 for performing chemical analyses of compounds that is analternative embodiment of the above-described method 100. The method 200includes digesting 110, separating 120, and storing 150 a profile of atleast one compound in the sample, as for the method 100.

For the processes of ionizing 130 and mass analyzing 140, the method 200includes the following described more detailed steps. These stepsprovide different processing for unknown proteins and for knownproteins. The different processing, as described below, providesreliable identification of unknown proteins in support of the definitionof correct profiles.

In response to a decision 231 that a protein of interest has an identitythat is known to an analyst, precursor ions are formed 235 from theknown protein, the precursors are mass analyzed 245, and the proteinprofile is then optionally defined and stored 150 using the knownidentity and the precursor-ion data associated with the observed valuesrelated to retention time, ion mass, and ion intensity.

Optionally, if product ions are formed from the known proteinprecursors, the product ions are mass analyzed, and the protein profileis then optionally defined and stored 150 using the known identity andthe product-ion data associated with the observed values related toretention time, ion mass, and ion intensity.

If the identity of the protein of interest in the sample is determined231 to be unknown to the analyst, both precursor and product ions areoptionally formed 236 from the protein, and both the precursor ions andproduct ions are mass analyzed 246. The product ions are used tovalidate identities of the precursor ions. The validated precursors, inturn, support reliable use of precursor data to identify the protein.For example, precursor data is used in a search of a pre-existingprotein database 242. One suitable procedure that is optionally used toimplement precursor validation is described in PCT Patent ApplicationInternational Publication No WO 2005/114930, published on Dec. 1, 2005,which is incorporated herein by reference.

Thus, if a polypeptide in the sample is an unidentified polypeptide, themethod 200 optionally includes identifying the polypeptide by searchinga pre-existing protein'database 242 for a protein having precursorsidentified via the analysis. Suitable databases 242 are commercially orfreely available, as known to one having ordinary skill in the proteinanalysis arts.

The database 242, for example, includes a list of proteins and, for eachlisted protein, associated tryptic peptides indicated by, for example,their precursor ion masses.

In one exemplary embodiment, the protein database 242 includes acollection of proteins and their theoretical peptide sequences. In thisexample, a protein in the sample is identified 241 by searching thedatabases' sequences for evidence of precursor and product ions thatcorrespond to a theoretical peptide sequence. If a sufficient number ofsuch masses are found in the data, and at a common retention time, thenthe peptide sequence is identified in the data. If, for example, thisapproach finds in the data one or more peptide sequences associated witha given protein, then the protein is taken to be identified 241 in thesample.

In some preferred implementations of the method 200, the data isgathered using an LC/MS system and a preselected database 242 (i.e., toaccomplish steps 120, 236, 246, 241.) For example, as described in moredetail with reference to FIGS. 9A and 9B, an eluent output by the liquidchromatograph is introduced into a mass spectrometer through anelectrospray interface. Optionally, a first quadrupole of atriple-quadrupole MS instrument functions as an ion guide. Analternating voltage is applied to a collision cell of the instrument.Spectra are collected of precursors ions and of their fragment (product)ions, for example in an alternating fashion, as described below.

Preferably, both precursor ions and associated product ions are formedfrom the same precursor material obtained from the separating 120process. In this manner, both precursor ions and associated product ionswill have the same retention time data determined from the separating120 process. Product ions may thus be relatively readily associated withthe precursor from which they arose. Less desirably, two or moreinjections of a sample are performed, and precursor-ion and product-iondata are obtained from different injections.

Any suitable method, including known methods, may be used to obtain bothprecursor and product ions from a single sample injection. Such methodsprovide effectively simultaneous mass analysis of both precursor andproduct ions. For example, a portion of an eluted precursor isfragmented to form product ions, and the precursor and product ions aresubstantially simultaneously analyzed 246, either at the same time or,for example, in rapid succession.

As an alternative example, two or more alternating portions of the peakare used respectively for precursor and product analysis. A portion of apeak's precursor material is ionized and analyzed, and then a nextportion is dissociated into product fragments that are analyzed. In onepreferred embodiment, alternating portions of an eluting precursor aresampled to alternately obtain data for the precursor ion and its productions. The obtained data permits reconstruction of a peak shape to permitmeasurement of an accurate retention time value for both the elutedprecursor and its associated product. Moreover, for example, peak shape,width, and/or time of reconstructed peaks associated with precursor ionsand with product ions are optionally compared to determine which productions are associated with a particular product ion.

One approach to such alternating, effectively simultaneous analysis, isdescribed in U.S. Pat. No. 6,717,130 to Bateman, et al. (“Bateman”),which is incorporated herein by reference and describes application ofan alternating voltage to a collision cell to regulate fragmentation.Additional description of related features is provided below withreference to FIGS. 9A and 9B.

Thus, the technique described in the Bateman or other suitable techniqueuses retention-time observations to support the determination of whichproduct ions are derived from a particular precursor. The product ionsare associated with their precursor ion in response to matchingretention-time values.

For example, a threshold retention-time difference is selected; if thedifference in retention times of a product ion and a precursor ion isless than the threshold value, the product is determined to be derivedfrom the precursor. For example, one suitable threshold value is equalto one tenth the retention-time peak width of the precursor ion. Theretention-time value of an ion is optionally defined as the time valueof the peak maximum of the peak that was observed for that ion.

Once a catalog of protein profiles is generated, as described above, thecatalog is optionally used to support accurate identification 170 ofproteins in subject samples without referring to a pre-existing proteindatabase. For example, a protein in a subject sample is optionallyidentified through a determination that the three most intenseprecursors of the protein—as listed in the profile of the protein—areappropriately present in the sample. The presence of the precursors inthe data is determined by matching the retention-time values, massvalues, and intensity-ratio values of the collected data with the valuesof the profile. Fewer or more precursors are optionally used todetermine matches and/or other than the three most intense precursorions may be utilized.

Use of the protein profiles provides a number of advantages over someprior identification methods. For example, a protein may be accuratelyidentified even when present at relatively low concentration. A lowconcentration sample often provides little or no detectability ofproduct ions. The corresponding protein profile, however, providesrelatively reliable, accurate precursor data for comparison if theprofile was developed from a sample having a relatively highconcentration of the protein.

After identification of the protein, the identification is optionallyconfirmed by comparisons of additional data for other precursors and/orcomparisons of product-ion data in the profile if expected to be presentat an observable level. The concentration of the protein in the subjectsample is optionally determined by comparisons to intensity-related dataof the profile.

A catalog of protein profiles is optionally supplemented and/or updatedupon analysis of additional reference and/or subject samples. Forexample, if the method 200 identifies a protein that is not present inthe catalog, a profile of the protein is defined and stored 150 in thecatalog. For example, if the method 200 observes a protein that alreadyhas a profile in the catalog, the existing data in the protein's profilemay be supplemented or replaced with the newly acquired data. Forexample, a more reliable profile is possibly obtained if the protein waspresent at a greater concentration in the newly observed sample thanexisted for the prior sample that was used to define the profile. Thus,both reference samples and subject samples are usable by an analyst tobuild and update a catalog of profiles.

For example, each injection provides a potential new profile for eachprotein present in that sample. The catalog is optionally continuouslyupdated, expanded and refined, as new injections are analyzed. Forexample, different samples may contain the same protein, but atdifferent concentrations. In general, the best available profile of aprotein is the one that has the highest intensities.

Thus, the analyst optionally commences with profiles obtained from afirst sample injection, and systematically compares these profiles toall profiles present in a (possibly large) catalog of previously definedprotein profiles. If the intensity associated with a profile in theinjection exceeds the intensity associated with a matching existingprofile in the catalog, then new data either supplements the data in theexisting profile, or simply replaces the existing data in the profile.

As described above, if a protein profile obtained from an injection isnot present in the catalog, then the protein profile is optionally addedto the catalog of profiles. Optionally then, an initial catalog is emptyand all subsequent injections are used to populate, refine, and/orexpand the protein profiles in the catalog. In some alternativeembodiments, a catalog includes one or more protein identities forprofiles that have yet to include precursor-related data. The profilesare then defined with data as the proteins are observed in analyzedsamples.

FIG. 3 is a flow diagram of another method 300 for chemical analysis,which includes providing 310 a catalog 332 of protein profiles obtainedthrough use of a pre-selected method and identifying 330 one or moreunidentified proteins in response to a comparison between the catalog332 and a determination 320 of intense precursor ions. If 340 the storedprofile has lower intensity values than provided by a newly observedprotein, the catalog is updated 350.

For example, one or more analysts develop the catalog 332 and one ormore analysts then utilize the developed catalog 332 to detect, identifyand/or quantify one or more proteins in a sample. The same or similarequipment is preferably used for sample analysis as was used to developthe catalog. The catalog can be obtained from similar equipment indifferent laboratories and generated by the same or different analysts.Catalogs obtained from the same or different equipment in the same ordifferent laboratories can be combined to form a single or additionalcatalogs.

As mentioned, protein-profile data is also optionally used to quantifythe concentration in a sample of a detected protein. For example, if theconcentration of the protein was known or determined for the priorsample that was used to define the protein's profile, the profileoptionally includes an indicator of concentration, where the indicatoris associated with the intensity values of the profile.

An indication of a concentration of a protein in a sample is obtained,for example, by comparing the most intense ion between profiles, or adesignated precursor and/or product ion, or designated sets of precursorand/or product ions. Comparisons of concentrations are made, forexample, by taking the averages or median values of intensities ofprecursors and products, and then taking the ratios of such averages ormedian values between proteins in different samples.

Optionally averages or median values of ratios of intensities areformed. Optionally, weighted or unweighted least-squares fitting is usedto scale intensities of one set of ions to match another; the scalingfactor obtained by least-squares fitting is used as an indication ofconcentration, or relative concentration of the proteins.

Next referring to FIG. 4, FIG. 5, FIG. 6, and FIG. 7, examples of someforms of protein profiles are described.

FIG. 4 is a three-dimensional graphical diagram of an example profileobtained from a sample of the protein transalodase (an enzyme) obtainedfrom E. Coli, according to one embodiment. The profile includes data forprecursor and product ions; the data are depicted in the graph as linesfor each ion, located according to mass and retention time of the ion.The height of each bar corresponds to the intensity (ion count) of theassociated ion. Precursor ions are identified by O's at the both ends ofa line and product ions are identified by X's at both ends of a line.The profile includes data for ten precursor ions and for more thanthirty product ions (not all of which are discernible in the diagram dueto similar mass, time, and intensity values.)

FIG. 5 is a two-dimensional bar graph of the profile of FIG. 4, showingintensity of precursor ions and product ions as a function of ion mass.As expected, the precursor ions generally have greater masses andintensities than the product ions since the product ions are fragmentsof precursors and more numerous.

FIG. 6 is a two-dimensional bar graph of the profile of FIG. 4, showingintensity of precursor ions and product ions as a function ofretention-time. The association between individual precursor ions andtheir product ions (i.e., same retention times) are observed.

FIG. 7 is a two-dimensional plot corresponding to a top-down view of theprofile of FIG. 4. The plot shows the location in mass and retentiontime of each precursor ion and product ion of the profile. Again, theassociation between individual precursor ions and their product ions areobserved due to their alignment along a common retention time.

Next referring to FIG. 8, some embodiments of the invention assistanalysis of relatively complex samples, such as those that have multipleproteins, which may have precursors with overlapping chromatographicpeaks. Some embodiments utilize sample fractionation to simplify and/orconcentrate components of a complex sample and/or provide multiplesample portions for analysis and comparison.

In some embodiments, a sample is fractionated to increase theconcentration of a protein in a fraction of the original sample. Theconcentrated sample is then optionally subjected to the above-describedanalysis methods 100, 200. Such embodiments increase the dynamic rangeof protein concentrations to which the methods 100, 200 areadvantageously applied.

For example, utilizing some methods of the invention, all proteinspresent in a cell's proteome or a serum proteome are potentiallyobservable for definition of protein profiles and/or identification ofthe proteins.

In some other embodiments, one of which is described with reference toFIG. 8, overlapping protein or precursor peaks are divided into two ormore portions; data obtained from the portions is compared to assist adetermination of which precursors are associated with the same protein.For example, after fractionation, the fractions are digested andseparated via a chromatographic process. Separated precursors whose peakintensities from fraction to fraction correspond to proteinconcentration from fraction to fraction are then associated with theprotein from which they arose.

By fractionating overlapping peaks of two polypeptides, one obtains asystematic change in ratio of subsequently produced precursors of thetwo polypeptides. LC/MS analysis of the fractions, such as byabove-described methods that preserve retention-time information,produce precursor ion data. Fraction-related data may then be comparedto determine which precursors are associated with a common polypeptide(in response to the different concentration ratio of the twopolypeptides in each fraction.) Thus, if retention-time data isinsufficient to determine the association, the additional informationprovided by fractionation often permits completion of the associationprocess.

Alternatively, for example, a digested complex protein sample issubjected to a chromatographic separation process; sample portions arecollected over a collection-time window that is preferably less than thefull width, of a chromatographic peak. Such a window ensures that atleast two fractions will each include portions of a protein.

FIG. 8 is a flow diagram of a method 800 for chemical analysis,according to one embodiment of the invention. The method 800 includesfractionating 810 a complex sample having at least two polypeptides,digesting 820 and performing 830 LC/MS on the first sample portion,digesting 840 and performing 850 LC/MS on the second sample portion, andassociating 860 precursor ions with their corresponding one of the firstand second polypeptides in response to ratios of the observedintensities of the precursor ions in the first and second sampleportions.

The complex sample is fractionated 810 into a least a first sampleportion and a second sample portion that each include portions of thetwo polypeptides though in different concentration ratios. The fractionsare digested 820, 840 via any suitable technique, such as thosedescribed above. LC/MS is performed 830, 850 to observe intensities ofprecursor ions associated with the polypeptides in the sample portions.Intensity related data then supports the determination of theassociation 860 of precursor ions with their polypeptide.

The method 800 is suited, in particular, to deconvolution of proteinsthat physically overlap during separation prior to fractionation 810.The method 800 is potentially most helpful in the case of two or morepolypeptides exhibiting overlapping peaks that have indistinguishableretention times. The method 800 may thus be utilized, for example, incombination with a method such as the above described methods 100, 200to generate a catalog of reliable profiles of proteins.

Any suitable fraction technique is used, including known techniques suchas gel-permeation chromatography, size-exclusion chromatography,ion-exchange chromatography, and reverse-phase chromatography. Somesuitable techniques utilize column overloading, pH gradients, and/ordenaturing mobile phases.

The fractions are optionally collected in, for example, a series ofvials. Alternatively, for example, each fraction is briefly held priorto subsequent analysis via, for example, a method such as theabove-described methods 100, 200.

Fractionation 810 is preferably performed by oversampling, that is, byemploying a fraction collection time of less than a peak width whencollecting fractions of overlapping peaks. For example, if a peak widthis 1 minute, a collection time of less than 1 minute is desirable.

Any suitable collection time value is selected. For example, onesuitable collection time value is about a full-width at half maximum(FWHM) of a peak. Such a collection time assures that at least twofractions will include portions of at least two overlapping peaks. Insome embodiments, three fractions are collected of overlapping peaks toprovide sufficient confidence in the subsequent association ofprecursors with sample polypeptides. More generally, a greater number ofnarrower collection windows are optionally used to provide a simplersample composition in each collected fraction.

Thus, a collection-window width is selected in any suitable manner. Awidth is optionally selected in response to an interactive process, inwhich a width that optimally assists deconvolution of peaks isdetermined. A single protein is optionally analyzed to empiricallydetermine a peak width. Some fraction of the determined peak width isthen selected for fraction collection, such as the FWHM of thedetermined peak. Alternatively, a peak width is determinedtheoretically.

In some alternative embodiments, proteins in a complex sample areconcentrated via fractionation; the fractions are subsequently digested,and the digested fractions themselves are fractionated via a peptidefractionation protocol. For example, the fractionated peptides, atpossibly a high column load, are fractionated by any one of a number ofwell-established protein fractionation techniques, such asgel-permeation chromatography, or reversed-phase chromatography, or ionexchange chromatography.

Each peptide fraction now represents a portion of the original peptidedigest. This fraction is optionally concentrated and injected on achromatographic column at a maximum column load. An LC/MS method usedfor all peptide digests is preferably the same (fractionated andunfractionated.) Thus, a given peptide will elute at the substantiallythe same retention time whether it is from the fractionated orunfractionated sample.

The fractionated peptides are then identified, for example, as describedabove. Any peptide seen in a fraction is seen at higher mass load withhigher intensity, and more of its fragment ions will be seen above thedetection limit of the instrument. Thus any high-energy validatedpeptide seen in the unfractionated digest will be seen in thefractionated digest with more ions, providing more sequence coverage.Peptides will also be seen in the fractionated digest with high-energyvalidation that were not seen in the unfractionated digest.

Retention time and accurate mass matching may be used to tie observedpeptides in the fractionated and unfractionated samples together. Ofparticular use is the ratio of intensity of a peptide precursor seen inthe fractionated sample to the precursor's intensity seen in theunfractionated digest. This ratio can be applied to all fragment ionsseen in the fractionated digest, and thus one can infer the intensity offragments as they would appear in the unfractionated digest. Thus onecritical feature of some alternative profiles, the measurement ofrelative intensities of peptide precursors to a protein is preserved.

Referring next to FIGS. 9A and 9B, some embodiments of the inventionrelate to LC/MS instruments. FIG. 9A is a block diagram of an LC/MSsystem 900, according to one embodiment of the present invention. Theinstrument includes a chromatography module 904 and a mass-spectrometermodule 912 that receives an eluent from the chromatography module 904.The LC module 904 includes an injector 106 that receives a sample 902, apump 908 and a column 910. the MS module 912 includes adesolvation/ionization device 914, an ion guide 916, a mass analyzer920, and a detector 922. The system 900 also includes a data storageunit 924 and a computer module 926.

In operation, the sample 902 is injected into the LC module 904 via theinjector 906. The pump 908 pumps the sample through the column 910 toseparate the mixture into component parts according to retention timethrough the column 910.

The output from the column 910 is input to a mass spectrometer 912 foranalysis. Initially, the sample is desolvated and ionized by thedesolvation/ionization device 914. Any desolvation technique can beemployed, including, for example, a heater, a gas, and a heater incombination with a gas or other desolvation technique. Ionization can beby any suitable ionization technique, including for example,electrospray ionization (ESI), atmospheric pressure chemical ionization(APCI), or other ionization technique. Ions resulting from theionization are fed to a collision cell 918 by the ion guide 916.

The collision cell 918 is used to fragment the ions. In preferredembodiments, the collision cell 918 is operated in a switching mode tosupport observation of both precursor ions and product ions of the sameeluting precursor material.

Any suitable switching techniques may be used, including knowntechniques. Some embodiments of the invention preferably use afragmentation protocol in which a relatively simple alternating voltagecycle is applied to the cell 918. This switching is done at a highenough frequency so that multiple high- and multiple low-energy spectraare contained within a single chromatographic peak. Unlike some otherswitching protocols, the cycle is independent of the content of thedata.

For example, as described in the '130 patent, an alternating voltage isapplied to the collision cell 918 to cause fragmentation. Spectra arecollected for the precursors (no collisions) and fragments (results ofcollisions.)

Alternative embodiments utilize other means for fragmentation, such asany suitable collision fragmentation or reaction device, including anysuitable known device. Some optional devices include: (i) a SurfaceInduced Dissociation (“SID”) fragmentation device; (ii) an ElectronTransfer Dissociation fragmentation device; (iii) an Electron CaptureDissociation fragmentation device; (iv) an Electron Collision or ImpactDissociation fragmentation device; (v) a Photo Induced Dissociation(“PID”) fragmentation device; (vi) a Laser Induced Dissociationfragmentation device; (vii) an infrared radiation induced dissociationdevice; (viii) an ultraviolet radiation induced dissociation device;(ix) a nozzle-skimmer interface fragmentation device; (x) an in-sourcefragmentation device; (xi) an ion-source Collision Induced Dissociationfragmentation device; (xii) a thermal or temperature sourcefragmentation device; (xiii) an electric field induced fragmentationdevice; (xiv) a magnetic field induced fragmentation device; (xv) anenzyme digestion or enzyme degradation fragmentation device; (xvi) anion-ion reaction fragmentation device; (xvii) an ion-molecule reactionfragmentation device; (xviii) an ion-atom reaction fragmentation device;(xix) an ion-metastable ion reaction fragmentation device; (xx) anion-metastable molecule reaction fragmentation device; (xxi) anion-metastable atom reaction fragmentation device; (xxii) an ion-ionreaction device for reacting ions to form adduct or product ions;(xxiii) an ion-molecule reaction device for reacting ions to form adductor product ions; (xxiv) an ion-atom reaction device for reacting ions toform adduct or product ions; (xxv) an ion-metastable ion reaction devicefor reacting ions to form adduct or product ions; (xxvi) anion-metastable molecule reaction device for reacting ions to form adductor product ions; and (xxvii) an ion-metastable atom reaction device forreacting ions to form adduct or product ions.

The output of the collision cell 918 is input to a mass analyzer 920.The mass analyzer 920 is any suitable mass analyzer, includingquadrupole, time-of-flight (TOF), ion trap, magnetic sector massanalyzers as well as combinations thereof. The detector 922 detects ionsemanating from the mass analyzer 920. The detector 922 is optionallyintegral with mass analyzer 920. For example, in the case of a TOF massanalyzer, the detector 922 is optionally a microchannel plate detectorthat counts intensity of ions, i.e., counts numbers of impinging ions.The storage medium 924 provides permanent storage for storing the ioncounts for analysis. For example, storage medium 924 is an internal orexternal computer disk. The analysis computer 926 analyzes the storeddata. Data can also be analyzed in real time without requiring storagein a storage medium 924. In that case, the detector 922 passes data tobe analyzed directly to computer 926 without first storing it topermanent storage.

The collision cell 918 performs fragmentation of the precursor ions.Fragmentation can be used to determine the sequence of a peptide andsubsequently lead to the identity of the originating protein.

The collision cell 918 utilizes a gas, such as nitrogen. When a chargedpeptide interacts with the gas' atoms, the resulting collisions canfragment the peptide by breaking it up at one or more characteristicbonds. The most common resulting fragments are described as Y- orB-ions. Such fragmentation can be accomplished as on-line fragmentationby switching the voltage in a collision cell between a low voltage state(low energy) which obtains MS spectra of the peptide precursor, with ahigh voltage state (high energy) which obtains MS spectra of thecollisionally induced fragments of the precursors. High and low voltageare referred to as high and low energy, since a voltage is used toimpart kinetic energy to an ion.

The chromatographic module 904 includes any suitable chromatographyinstrument(s), including known instruments, such as column-basedinstruments. Suitable columns include columns known to one havingordinary skill in the chromatographic arts. The column can be formedfrom, for example, metallic or insulating materials. Suitable materialsinclude known materials such as steel, fused silica, or lined materials.The column can include more than one column, disposed in serial and/orparallel configurations. For example, the column can be a capillarycolumn and can include multiple capillary tubes.

The computer module 926 is in data communication with other componentsof the system 400 via wired and/or wireless means, such as those knownin the data-communication arts. The module 926 receives process data,for example, from the mass-spectrometer module 912, and provides controlsignals. The module 926 is optionally configured to implement methodsdescribed herein, such as the methods 100, 200 for chemical analysisdescribed above. The module 926, in various illustrative embodiments, isimplemented in software, firmware, and/or hardware (e.g., as anapplication-specific integrated circuit), and includes, if desired, auser interface. The module 926 includes and/or is in communication withstorage component(s), such as the storage unit 924.

Suitable implantations of the module 926 include, for example, one ormore integrated circuits, such as microprocessors. A single integratedcircuit or microprocessor in some alternative embodiments includes themodule 926 and other electronic portions of the system 900. In someembodiments, one or more microprocessors implement software that enablesthe functions of the module 926. In some embodiments, the software isdesigned to run on general-purpose equipment and/or specializedprocessors dedicated to the functionality herein described.

An LC/MS experiment can produce as one of its outputs a masschromatogram. A mass chromatogram is a set or group of responses(intensities) recorded as a function of time at a specific mass value.In a mass chromatogram, the mass value may be the central value within arange. That is, the intensity at a given time may be obtained bycombining intensities collected over a specified range of mass values.Typically, a mass chromatogram contains one or more chromatographicpeaks.

A single molecule, or chemical entity, has a specific mass. In an LC/MSexperiment the ionized form of that molecule is observed as achromatographic peak at the mass value of that ion divided by its charge(mass-to-charge ratio). A chromatographic peak has a peak profile, orelution profile. The chromatographic peak profile can be characterizedusing several features, including an apex retention time, a peak width,a lift off time and a touch down time. A chromatographic peak width canbe described as a width at a specific peak height (FWHM, width at 50%height), or a width between inflection points, or as a standarddeviation. The apex intensity or chromatographic peak height is themaximum intensity found in a chromatographic peak profile. Generally,the apex intensity is baseline corrected.

A molecule in an eluent that is separated by a chromatographicseparation, and elutes from the column is referred to as the commoneluting molecule or originating molecule. The originating molecule isionized through the ionization source of the mass spectrometer. Theresulting ions are measured in an LC/MS or LC/MS^(E). As a result ofisotopic composition and or fragmentation processes, each originatingmolecule can give rise to multiple categories of ions, each having aunique value of mass and charge. The ion corresponding to theoriginating molecule is termed the precursor ion, or just the precursor.

In peptide digests the originating molecule is a peptide and the ioncorresponding to the peptide is referred to as the precursor. Any ionderived from the originating molecule, whether the processor or afragment, must have the same retention time and chromatographic peakprofile as the precursor.

In an LC/MS experiment an ion can be described and/or referred to by itsretention time, mass-to-charge ratio, and intensity. A single moleculecan appear in an LC/MS chromatogram as a cluster of ions. A peptidegives rise to one or more ion clusters. Each cluster corresponds to adifferent charge state (e.g., Z=1 or Z=2). Each ion in a clustercorresponds to a different isotopic composition of the peptide. In acluster of ions from a common peptide, the monoisotope is the ion havingthe lowest mass, where all the isotopes are in their most abundant, lowmass state. Since the ions in the cluster come from a common originatingmolecule, they must share a common retention time and peak profile.

An originating molecule can give rise to multiple ions due to isotopeand charge effects. Additional, important sources of ions are fragmentsof the originating molecule. These fragments arise from processes thatbreak up the originating molecule. These processes can occur in theionization source or in a collision cell. Because fragment ions derivefrom a common eluting, originating molecule, they must have the samechromatographic retention time and peak profile as the originatingmolecule.

Generally, if an originating molecule gives rise to N ions, and if theseare adequately resolved by the mass spectrometer, then there can be Nmass chromatograms, where each mass chromatogram contains a peak, achromatographic profile of an ion that derives from the originatingmolecule. The retention time and peak profile of each of these N ionswill be identical. The term common-retention-time-entity refers to allions of an originating molecule that, in an LC/MS separation, give riseto chromatographic peaks all having the same retention times and peakshapes.

The retention time and peak shapes of ions that derive from a commonoriginating molecule are the same because the time of ion formation,fragmentation, and ion detection is generally much shorter then the peakwidth of the originating molecule. For example, a typicalchromatographic peak width, measured at full-width at half-maximum(FWHM) is 5 to 30 seconds. The time of ion formation, fragmentation, anddetection is typically sub milliseconds. Thus on a chromatographic timescale, the time of ion formation is an instantaneous process. It followsthat differences in observed retention times of the ions that derivedfrom an originating molecule is effectively zero. That is,sub-millisecond retention time differences between ions that derivedfrom an originating molecule are small compared to the chromatographicpeak width.

The ions that are associated with an originating molecule fall into oneof several categories. An ion derived from an originating molecule canbe a precursor, a fragment of the precursor, or a fragment of afragment, or a neutral loss of any of the above masses. Any of thesemasses can be seen in one or more discrete isotopic states, and in oneor more charge states.

In the case of peptides, a given peptide is generally seen to be acluster of ions, each in a distinct isotopic state, and each in one ormore charge states. Ideally the ionization source produces precursorsthat are a protenated form of the neutral originating molecule. One ormore protons can be attached to the neutral molecule and thus theprecursors can be one or more mass units higher than the neutral withcharge Z=+1, or +2, etc. In practice, this precursor (termed mwHPlus)may be accompanied by lower mass entities that result from the loss ofneutral molecules such as water, ammonia, or phosphate. Fragmentationcan occur in the source, yielding, typically, Y- or B-ions.Fragmentation can be also be deliberately induced by down-streaminteractions with gas molecules in a collision cell.

With respect to ions that are generated from collision-induceddisassociation of intact precursor ions, the fragment product ionsassociated with their parent precursor ion. This association isaccomplished without requiring the instrument to pre-select a singleprecursor for subsequent fragmentation using the mass spectrometer in aHigh-Low Data Acquisition Mode. More specifically, associated ions areappropriately grouped when multiple precursors are fragmentingsimultaneously, at essentially the same retention time. Thus,embodiments of the present invention can assign product ions to theirrespective precursor when there is more than one precursor fragmentingat the same moment in time. Moreover, embodiments of the presentinvention can significantly reduce the computational burden forde-isotoping and charge-state reducing ions to their common, singlycharged annotation (i.e. MH+).

The method of the current invention can be applied to mixtures otherthan that of peptides, provided originating molecules give rise toprecursor ions and fragment ions. Thus embodiments of the presentinvention can be used in proteomics, metabolomics, and metabonomics.

The retention time and chromatographic peak profile of a molecule(peptide, metabolite, natural product) eluting from a chromatographicsupport matrix, such as column 910, is a function of the physicalinteraction of that molecule between the support matrix and mobilephase. The degree of interaction that a molecule has between the supportmatrix and the mobile phase dictates the chromatographic profile andretention time for that molecule. In a complex mixture, each molecule ischemically different. As a result, each molecule can have a differentaffinity for the chromatographic matrix and the mobile phase.Consequently, each can exhibit a unique chromatographic profile.

Generally, a chromatographic profile for a specific molecule is uniqueand describes the physicochemical properties of that molecule.Parameters optionally used to characterize the chromatographic peakprofile of a given molecule include the time of initial detection(liftoff), normalized slope, the time of inflection points relative tothe time of the peak apex, the time of maximum response (peak apex), thepeak width, at inflection points, at full-width-at-half-maximum (FWHM),peak shape asymmetry, and the time of the final detection (touch down)to name only a few.

FIG. 9B shows three related graphs that illustrate the collection ofmass spectra during a period of time that covers an eluted peak of aprecursor, according to one embodiment of the invention. A first graph994 illustrates the alternating collection over elution time oflow-energy spectra (i.e., spectra from unfragmented precursors, labeled“MS”) and high-energy spectra (i.e., spectra from fragmented precursors,that is, product ions, labeled “MS^(E)”.) Second and third graphs 994A,994B respectively illustrate the MS and MS^(E) spectral collection timesand the reconstruction of the retention time peak associated with theprecursor.

The reconstructed peak represents the chromatographic elution profile ofa single precursor. The horizontal axis corresponds to elution time ofthe peak profile. The vertical axis corresponds to arbitrary units ofintensity associated with the time-varying concentration of theprecursor as it elutes from the chromatographic column.

An eluting precursor, passed to the mass spectrometer, thus producesions in both low- and high-energy modes. The ions produced in thelow-energy mode are primarily those of the precursor ions in possiblydifferent isotopic and charge states. In proteomic studies, theprecursor ions are peptides generated from enzymatic digestion(typically a tryptic digest) of the intact protein(s). In high-energymode, the ions are primarily different isotopes and charge states of thefragment ions of those precursors. High-energy mode can also be referredto as elevated-energy mode.

In the graph 994, the alternating white and black bars thus representthe times at which spectra are collected with low and high-energyvoltages of the eluting chromatographic peak. The low-energy graph 994Adepicts the times at which a low-energy voltage is applied in thecollision cell 918, resulting in low-energy spectra. The high-energygraph 994B depicts the times at which a high-energy voltage is appliedin the collision cell 918, resulting in high-energy spectra.

The chromatographic peak of the precursor is thus sampled multipletimes, by the high- and low-energy modes. From these multiple samples,accurate retention times of all the ions associated with the peak andseen in the high- and low-energy spectra can be inferred. These accurateretention times are obtained by interpolation of the intensities sampledby the respective spectra.

What will now be described are techniques that may be used in connectionwith performing retention time matching of precursors and relatedproduct ions included in an input data set.

In connection with a sample or mixture, such as a complex protein sampleincluding multiple proteins, many precursor ions may have a sameretention time. When a precursor ion is fragmented, the product ionsproduced as a result of the fragmentation will also have the sameretention time as that precursor. Due to the large number of precursorions that may have the same retention time, product ions from differentprecursors may have substantially the same retention time. As a result,it may be difficult to match product ions to the respective, correctprecursor ions. The matching of product ions to the appropriateprecursor ion from which the product ions are generated has manyapplications as described herein and known to those skilled in the art.

In the context of LC/MS, the retention time matching technique findsthose product ions and the related precursor ion from which the productions are derived having the same retention time and peak shape. Thetechniques described herein provide for association of product ions withprecursors ensuring that product ions and precursors havingsubstantially the same measured retention time are included in theoutput spectrum based upon retention time alignment.

The techniques for performing retention time matching may be used inconnection with complex samples as well as simple samples. Complexsamples may include, for example, a protein mixture as well as any oneof a variety of different biological samples known in the art such as aserum, tissue, and cells. The retention time matching techniques mayalso be used in connection with a simple sample of a single polypeptide.

The techniques for retention time matching of precursors to relatedproduct ions may be used, for example, in connection with the techniquesdescribed herein to produce a polypeptide profile used in proteinidentification techniques. In connection with such a profile for aprotein as described herein, a set of precursor ions determined as themost intense precursors for the protein may be used to identify theprotein. The profile may be used to detect, identify, track and/orquantify the protein to a sufficient specificity so that the protein maybe distinguished from other proteins. The profile may also includeadditional information regarding each of the most intense precursors.The additional information may include, for example, one of more productions associated with each of the precursors, data (e.g., such asretention time, intensity and/or mass or m/z) about each of the one ormore product ions. The retention time matching techniques may be used toidentify the product ions associated with the most intense precursors asincluded in the profile. Information from the profile, such as the massof the most intense precursor ions along with the masses of a sufficientnumber of its product ions, can identify the sequence of the protein toa high degree of confidence.

The retention time matching techniques described in following paragraphsmay be used to detect, identify, track and/or quantify peptides andproteins and addressing problems in proteomics. The retention timematching techniques described herein may also be used in connection withsamples or mixtures that may be characterized as other than biological.In connection with proteomic applications, the peptides may result fromenzymatic digestion of sample proteins. Reliable identification ofpeptide precursors allows identification and quantitation of sampleproteins.

The retention time matching techniques described herein may be used toreliably assign or match product ions to precursors in a deterministicmanner without utilizing other methodologies, such as statisticalmethods, to compensate for incorrect matching of product ions andprecursors. The precursors and related product ions identified using thetechniques herein may be stored in a database alone, or in connectionwith other data such as when annotating an existing data store.

Although reference in illustrative examples herein may be made toapplications using protein digests analyzed using the foregoingtechnique described in Bateman, an embodiment may produce data setsusing other methodologies known in the art such as, for example, datadependent analysis (DDA) used to isolate selected precursor ions andidentify product ions for the selected isolated precursor. In oneembodiment, a mass spectrometer may be used to perform DDA in which themass spectrometer includes a collision cell and a quadrupole. Whenoperating in accordance with the DDA technique, the quadrupole is usedas a filter in a first phase to selectively isolate and select onlyprecursors of interest. Thus, only selected precursors are produced asan output of the first filtering phase. The selected precursors are thenpassed to a collision cell where they are fragmented, as using asufficiently high voltage, to generate fragments or product ions andobtain a desired number of scans for the isolated precursor and productions. The foregoing DDA technique may be repeated for isolatingdifferent precursors and obtaining a desired number of scans for theprecursors and related product ions.

An embodiment may determine masses of particular precursors of interestusing a variety of different techniques. For example, in one embodimentutilizing the Bateman techniques as described elsewhere herein, the lowenergy (LE) cycle or mode may be used to generate spectra including oneor more precursor ions. Other techniques used to generate the input dataset, such as the DDA technique, may also be used to isolate precursorsand determine their particular masses. The selected precursors andassociated masses may be subsequently identified in the input data set.

In one embodiment using the techniques herein, mass spectra as producedfrom different experiments using a mass spectrometer may be compared.The retention time matching techniques described herein may include themass spectra in an input data set, and may combine the foregoing massspectra by determining an intersection of the mass spectra. Forsimplicity of illustration and explanation, the mass spectra may includedata related to a single precursor and product ions having substantiallythe same measured retention time and peak shape as the precursor.However, the product ions have different mass or m/z values. Theretention time of the single precursor and its related product ions ineach of the different spectra may be within an expected retention timewindow of error incurred due to possible measurement error. In oneembodiment, the window of error may be within a threshold of 1/10^(th)of a peak width of the retention time of the precursor as determinedusing the full width half maximum (FWHM) methodology. As known in theart, FWHM is determined as the distance between two points on eitherside of the mass spectral peak at which the curve reaches half itsmaximum value. An embodiment may also use other values as the foregoingwindow of expected error in accordance with the expected error of systemand methodologies utilized in an embodiment.

The mass spectra included in the data set may include precursors havinga retention time within the foregoing retention time window or window oferror. Each of the mass spectra may then be aligned or normalized inaccordance with a single retention time. For example, the mass spectrain the data set may include those mass spectra having a precursor with aretention time of “n”+/− 1/10^(th) the chromatographic FWHM of the massspectral peak. Each of the spectrum in the data set may then be alignedat a single retention time, such as “n”. In the alignment process, eachprecursor ion in a spectrum is shifted by a quantity and in a directionto align the precursor at a retention time of “n”. Additionally, theproduct ions of the spectrum are also shifted by the same quantity andin the same direction in accordance with the shift of the precursor ofthe spectrum. The foregoing alignment is repeated for each of thespectrum. After alignment, if a product ion falls within the foregoingwindow of error in each of the spectra in the data set, then thatproduct ion is determined to also have the same retention time as theprecursor and is matched with the precursor for which the alignmentprocess is performed with the retention time of “n”. In contrast, if theproduct ion is not within the foregoing window of error for each of thespectra, the product ion is determined to be not a match for theprecursor. As such, the retention time matching of precursor and relatedproduct ions may be performed in a deterministic, reliable manner. Theresult may be in the form of an output spectrum containing the precursorand all identified product ions from the mass spectra of the input dataset.

The data set may include spectra, such as MS spectra, generated using avariety of different techniques. For example, the spectra may beobtained using an LC/MS analysis of complex mixtures using thetechniques of Bateman or the DDA technique. The data set may also beobtained from MALDI-MS-MS, and using spectrometers with high or lowresolution.

The product ions as included in a data set for use in connection withthe retention time matching techniques may be produced using a varietyof different methodologies known in the art. The product ions may beproduced using any one of a variety of different fragmentationtechniques. An embodiment may use a mass spectrometric (MS) methodologyas described in Bateman using a high- and low-energy switching protocolapplied as part of an LC/MS analysis of a single injection of a peptidemixture. In such data the low-energy (LE) spectra contain ions primarilyfrom unfragmented precursors, while the high-energy (HE) spectra containions primarily from fragmented precursors.

Each spectrum in the data set to which the retention time matchingtechniques described herein are applied may be obtained from anindependent analysis or experiment. For example, in an LC/MS context,each of the M spectra included in an input data set may be obtained fromM different injections. These M injections may be from M injections ofthe same aliquot (e.g., replicate injections). Alternatively, each ofthe M injections may use a different sample mixture. An embodiment mayalso utilize a data set in which the spectra are produced from somenumber of replicate injections of some number of different samplemixtures.

The retention matching technique depends on the principle that productions maintain strict association with the precursor ion from which theproduct ions are derived. This association may manifest itself by boththe product ions and the precursor ion appearing at substantially thesame measured retention time. The retention matching technique takesadvantage of the fact that ions which are unrelated to a selectedprecursor will not maintain the foregoing association for the spectraanalyzed in the input data set.

The retention time matching technique makes use of the mathematicaloperation of Venn intersection as applied to masses of the ions. Twoions, such as a precursor and a product ion, are deemed to be related iftheir masses lie within a predetermined mass tolerance window and eachhave a same retention time as determined in accordance with the errorwindow size or retention time window as described elsewhere herein. Inan embodiment, the error window size or retention window used inconnection with matching a precursor with a product ion, may be relatedto the chromatographic FWHM of the mass spectral peaks, or othertolerance as related to the resolution of the instrument, such as the MSinstrument used to obtain the spectra in the input data set.

It should be noted that as described herein, two ions may be deemed tohave a same mass if a first mass of the first ion is within apredetermined mass tolerance of a second mass of the second ion. Thismass tolerance may be used in connection with the techniques describedherein with respect to precursor ions as well as product ions. In oneembodiment, the mass tolerance may be +/− 1/10^(th) of the FWHM of thepeak as may be included in a mass spectrum expressed inparts-per-million (PPM). Other mass tolerances may be used in connectionwith, and may vary with, an embodiment.

As a result of using the retention time matching technique describedherein, an output spectra may be produced which includes those productions deemed to be related to a precursor ion.

The techniques for retention time matching using Venn intersection maybe applied to M spectra such that a product ion is included in theoutput spectrum only if it appears in all M spectra within thepredetermined window size. Alternatively, an embodiment may apply theretention time matching using the Venn intersection such that a production is included in the output spectrum if the product ion appears in aspecified portion of the M spectra within the predetermined window size.The portion size selected may vary with an embodiment.

The retention time matching techniques described herein may be applied,for example, to spectra in which the number of unrelated ions greatlyexceeds those product ions that are related to a given precursor. Thesetechniques may be used to simplify the spectra that, taken individually,may be characterized as too complex to be interpreted using existingdata interpretation techniques.

As one example, a spectrum may include 4 product ions that are derivedfrom a common precursor peptide. The foregoing 4 product ions may beincluded in a spectrum also including 200 other product ions which areunrelated to the precursor peptide. The retention time matchingtechniques described herein may be applied to multiple spectra,including the foregoing four product ions and other unrelated productions. Ions unrelated to the precursor will not be included in the outputspectrum produced as a result of applying the retention time matchingtechniques. For example, the retention time matching techniquesdescribed herein may be used in connection with an input set includingthree spectra in which the foregoing 4 product ions related to thecommon precursor may be identified as such. The number of spectra neededfor reliable matching may vary with precursor and other factorsparticular to an embodiment. Given the precursor mass and the fourmasses of the related product ions found in the simplified outputspectrum produced as a result of the techniques herein, a search enginemay be used in identifying a peptide. Such search engines may include,for example, MASCOT from Matrix Sciences. As another example, thetechniques described herein may utilize an input data set including asfew as two input spectra. Each of the input spectrum may include aprecursor of interest and as few as a single product ion associated withthe precursor of interest.

In the context of LC/MS, the retention time matching technique findsthose product ions and the related precursor ion from which the productions are derived having the same retention time and peak shape. Thetechniques described herein provide for association of product ions withprecursors ensuring that product ions and precursors havingsubstantially the same measured retention time are included in theoutput spectrum based upon retention time alignment.

In addition to spectra, an input data set used in connection with thetechniques described herein may include ions in an ion list. An ion listmay be obtained, for example, from three-dimensional data such as may beacquired utilizing LC/MS or other experimentation and processingmethodologies. Each ion included in an ion list may be annotated by theion's retention time, mass or m/z, and/or intensity. In such instanceswhere three-dimensional data is utilized, spectra may be obtained, forexample using retention time binning as described, for example, in Plumbet al., US Patent Publication No. 2005/0127287, filed on Nov. 16, 2004,titled Method of Using Data Binning in the Analysis ofChromatography/Spectrometry Data, which is incorporated by referenceherein, or PCT Patent Application No. PCT US2005/004180, filed on Feb.11, 2005, titled Apparatus and Method for Identifying Peaks in LiquidChromatography/Mass Spectrometry Data and for Forming Spectra andChromatograms, by Gorenstein et al.

The retention time matching techniques described for matching precursorand product ions using the Venn intersection may be applied to a varietyof different areas and used in connection with a variety of differentmethodologies. For example, these techniques may be used in proteomicsand small molecule studies. These techniques may be used to detectprecursor and related product ions in replicate injections of a sampleand the storage of such information in the database, such as anannotated peptide catalog and included in a peptide profile. Such storedinformation may be extracted from a data store for comparison againstcharacteristics of unknown samples. Such stored information may be usedto detect, identify, and/or quantify an unknown sample.

In connection with the techniques described herein, multiple precursorshaving a same retention time in one injection are found to have slightlydifferent retention times and other injections even under replicateconditions, for example, as may be determined utilizing the Batemantechnique. Accordingly, product ions associated with multiple precursorsmay have a single retention time in the first injection and the multipleprecursors may have slightly but measurably different retention times inother injections. As a result, the product ions that may have a firstretention time in a first injection may have a slightly differentretention time in a subsequent injection even under replicateconditions. The techniques described herein advantageously utilize thefact that as long as the difference in measured retention time betweenthe precursor and the product ions are within the specified retentiontime window of error or threshold for a specified number of experiments,then the product ions may be associated with the precursor. Furthermore,the techniques described herein, utilize the fact that unrelated productions will not remain within the foregoing retention time window orthreshold for the specified number of experiments. Thus, the foregoingtechniques for retention time matching provide for separating productions which are related from those which are unrelated with respect to aprecursor.

It should be noted that the foregoing techniques compare mass values ofspectral peaks within the input data set or spectrum. No prior knowledgeregarding mass values or m/z values for the precursor and/or productions is needed. Additionally, no prior knowledge of the sequence for agiven protein is needed to utilize the techniques described herein on asample although the techniques described herein may be used to furtherannotate a database or catalog.

The output produced as a result of the techniques described herein maybe in the form of a spectrum. The spectrum may include the precursor andthe one or more product ions included as a result of determining theVenn intersection. The resulting output spectrum may be stored,displayed, used in connection with searching to identify an unknownpeptide, retention time tracked, used in connection with intensityratios between different injections, and the like. In one embodiment,multiple output spectra may be determined for the same pairing ofprecursor and product ions. For example, a first experiment maydetermine a first output spectrum having a first intensity. A secondexperiment may be performed for the same sample having a differentintensity under replicate conditions producing a second output spectrum.The output spectrum having the greater intensity may be stored.

Referring now to FIG. 10 and FIG. 11, shown are flowcharts of processingsteps that may be used in an embodiment in connection with performingretention time matching of a precursor and its related product ions. Thesteps of FIG. 10 and FIG. 11 summarize processing just described. Atstep 1002, an input data set is obtained. As described herein, the inputdata set may include data from multiple injections. The input data setmay be in any one or more of a variety of different forms including ionlists and spectra although spectra are included in the steps offlowcharts 1000 and 1050 for illustration. At step 1004, a firstspectrum of the input data set is selected. The first spectrum may bereferred to as the reference spectrum. The injection which was used inobtaining the first spectrum may be referred to herein as the referenceinjection. A precursor for the reference spectrum is determined. In oneembodiment, one or more precursors may be determined as those ionshaving the greatest mass and intensity. For purposes of illustration inconnection with the flowcharts 1000 and 1050, it is assumed that only asingle precursor is included in each spectrum of the input data set. Asdescribed herein, such as in connection with the Bateman technique,different methodologies may be utilized to determine the mass of aselected precursor of interest. With reference to the Bateman technique,masses of precursors may be determined by examining the resultingspectra produced using a LE scan.

In this first spectrum or reference spectrum, the product ions havingthe first retention time within the expected retention time error windowor threshold may be characterized as product ion candidates for theprecursor. After performing processing of the common retention timematching technique described herein, it is known which of the production candidates are matched or associated with the precursor ion.

When determining the product ions of the reference injection occurringat the same retention time as a precursor of the reference injection,all product ions occurring within the expected error window of theretention time are considered. For example, a precursor may have aretention time of T1 in the reference injection. A first product ion mayhave a retention time which falls within T1 and T1+/− the expected errorwindow or retention time window. The first product ion is considered asa candidate product ion for the precursor. If the first product ion hasa measured retention time which is outside the range of T1+/− theexpected error window or retention time window, then the first production is not considered as a candidate. The foregoing expected errorwindow is also used in subsequent processing steps in connection withmatching product ions to precursors in target injections when performingVenn intersection processing described herein. A target injection mayrefer to an injection of the input data other than the referenceinjection. Target injections may be used in producing the remainingspectra processed in the flowchart 1000 in the loop formed with a toptesting step 1006.

At step 1006, a determination is made as to whether all the spectra inthe input data set have been processed. If not, control proceeds to step1010 where the variable current spectrum is assigned to the nextspectrum in the input data set. At step 1012, the precursor and productions for the current spectrum are determined. The current spectrum issearched for an ion having the same mass and retention time (within asecond threshold or window) as the precursor of the reference spectrum.It should be noted that the product ions present in the referencespectrum also appear in the current spectrum.

An embodiment may also utilize the foregoing second threshold or windowrepresenting a window of time used when searching for a precursor havinga particular mass in a target injection such as in step 1012 processing.For example, a precursor having a mass m1 at a retention time T1 may bedetermined in a reference injection. For a subsequent target injection,processing searches for an ion having the same mass m1 and a retentiontime of T1+/− the second window or threshold. The second threshold orwindow may be empirically determined and may vary in accordance with anembodiment. For example, an embodiment may assign an initial value tothe second threshold, such as based on 2-3 chromatographic peaks widths.The second threshold may be modified or refined in accordance withempirical experimentation of a system. For example, if an embodimentutilizes a system or methodology introducing a large amount of error ornoise, the second threshold or window may be increased.

In connection with step 1012 processing, it should also be noted that aprecursor in the current spectrum is identified as being at a mass andis matched to the precursor in the reference or first spectrum of step1004 having the same mass. A first mass of the precursor in the firstspectrum may be deemed to be the same mass as a second mass of aprecursor in the current spectrum if the first mass is within thespecified mass tolerance of the second mass.

At step 1014, the precursor of the current spectrum may be time-alignedwith the precursor of the reference spectrum and all product ions in thecurrent spectrum are appropriately and accordingly time shifted. Forexample, if the retention time of the precursor in the referencespectrum is 10.0 minutes and the retention time of the precursor in thecurrent spectrum is 9.8 minutes, the precursor and product ions in thecurrent spectrum are shifted +0.2 minutes. Once the shifting iscomplete, control proceeds to step 1020 where the product ions in thecurrent spectrum which are within the expected retention window of erroror threshold tolerance are determined. At step 1020, the particularproduct ions which are within the expected window of error may berecorded for use in a later processing step.

It should be noted that the processing steps of flowcharts 1000 and 1050set forth steps that may be used in specifying a predetermined number orportion of the spectra that a product ion must be included in to qualifyas matching the precursor. The predetermined number or portion may beequal to, or less than, the total number of spectra in the input dataset. For example, as described herein, an embodiment may specify athreshold or minimum number of spectra less than the total number in theinput data set. If a candidate product ion is included in at least thespecified threshold number of spectra within the expected window oferror for a retention time of a precursor, then the candidate production may be determined as being associated with the precursor.

Control then Proceeds from Step 1020 to Step 1006.

If the determination at step 1006 evaluates to yes, control proceeds tostep 1051. In step 1051, a list of unique product ions in accordancewith the predetermined mass tolerance window is determined. The productions included in the first or reference spectrum and subsequent spectrumof the input data set as determined at step 1020 are examined. A firstproduct ion in one spectrum having a first mass may be deemed as havinga same mass as another product ion in a second spectrum if the firstmass and the second mass are within the predetermined mass tolerancewindow. In connection with the techniques described herein, the firstand second product ions may be deemed to be the same product ion in twodifferent spectra. From step 1051, control proceeds to step 1052. Foreach product ion as determined in step 1051, the number of spectra fromthe input data set in which each product ion is determined as having aretention time of the precursor within the retention time window oferror is tabulated. At step 1054, the current product ion variable isset to the first product ion. At step 1056, a determination is made asto whether the product ion has substantially the same retention time asthe precursor (e.g., within the retention time window of error) for thethreshold or predetermined number of spectra. In other words, if anembodiment specifies a threshold number of spectra at “M”, it indicatesthat a product ion has to have a same retention time as the precursorwithin the retention time window for “M” spectra in order for step 1056to evaluate to yes. If step 1056 evaluates to yes, control proceeds tostep 1058 where the product ion is determined to be associated ormatched with the precursor. From step 1058, control proceeds to step1062. If step 1056 evaluates to no, control proceeds to step 1060 wherethe product ion is determined not to be associated or matched with theprecursor.

Control proceeds to step 1062 where a determination is made as towhether all the candidate product ions have been processed. If so,control proceeds to step 1064 to generate an output spectrum. Asdescribed elsewhere herein, the output produced may be in a form otherthan a spectrum, such as an ion list. The output spectrum or otheroutput generated in step 1064 may include the product ions determined tobe associated with the precursor by having substantially the sameretention time and peak shape as the precursor. As described herein, theproduct ions are candidates which have been determined in accordancewith criteria including a threshold number of spectra from the inputdata set. If a candidate product ion is determined to have a retentiontime within a retention time's expected window of error for thethreshold number of spectra, the candidate product ion is deemed to havethe same retention time as a precursor with the retention time. If step1062 evaluates to no, control proceeds to step 1066 where the currentproduct ion variable is assigned to next product ion to be examined.

The techniques described herein will now be illustrated with additionalfigures. For the sake of simplicity of illustration, only a singleprecursor is illustrated although the techniques described herein may beused in connection with samples in which multiple precursors have acommon retention time.

Referring now to FIG. 12, shown is a graphical representation of areference injection. In this example 1100, a precursor 1102 and productions 1102, 1104, 1106, 1108, 1110 and 1112 have the retention time of1102 as indicated by 1114. It should be noted that, all ions having ameasured retention time within the retention time window of expectederror are considered.

Referring now to FIG. 13A, shown is a graphical representation ofanother injection. The injection of the example 1120 may be referred toas a first target injection. In this example 1120, the data for thetarget injection is searched for a retention time for the same mass asthe precursor from the reference injection. The search for the retentiontime is performed with respect to the second threshold or predeterminedwindow as described above. In the example 1120, the precursor 1102 isillustrated as having a retention time indicated by 1124. In FIG. 13B,shown is the alignment or normalization of the first target injection inaccordance with the retention time of the precursor from the referenceinjection. The precursor from the reference injection and the firsttarget injection are aligned. The product ions are also accordinglyshifted. It should be noted that as described for the referenceinjection, the precursor of a target injection may be determined asthose one or more ions having the greatest mass and intensity. Inconnection with the example 1124, note that product ions 1104 and 1106do not have the same retention time of 1114 as the precursor. Theremaining product ions 1108, 1110 and 1112 have the retention time 1114within the expected window of error of the retention time.

Referring now to FIG. 14A, shown is a graphical representation of asecond target injection. In this example 1140, the data for the secondtarget injection is searched for a retention time for the same mass asthe precursor from the reference injection in order to determine aprecursor ion. The foregoing searching to determine the precursor ion isperformed in a manner similar to as described in connection with FIG.13A. In the example 1140, the precursor 1102 is illustrated as having aretention time of 1142 and substantially the same mass (e.g., within themass tolerance) as the precursor 1102 in FIG. 13A. In FIG. 14B, shown isthe alignment or normalization of the second target injection inaccordance with the retention time of the precursor from the referenceinjection. The precursor from the reference injection and the secondtarget injection are aligned at retention time 1114 and each of theproduct ions is also accordingly shifted. In the example 1144, note thatproduct ion 1106 is illustrated as not having the retention time 1114within a retention time window of expected error as may be determined,for example, using +/− 1/10^(th) the FWHM of the mass spectral peak asdescribed elsewhere herein.

An example of an output spectrum including the precursor and associatedproduct ions as determined using the techniques described herein isillustrated in FIG. 15.

Based on the foregoing examples, it may be determined that only productions 1108, 1110 and 1112 are matched or associated with the precursorion 1102 having a retention time illustrated by 1122. Product ions 1104and 1106 were not within the retention time window of expected errorwith respect to 1114 in the first target injection. Product ion 1106 wasnot within the retention time window of expected error with respect to1114 in the second target injection.

The foregoing techniques for performing retention time matching may beused to annotate a database or catalog, such as a peptide catalog. Asknown in the art, for example, a protein sequence database may beinitially obtained and stored on a data storage device. The database maybe annotated using the techniques just described. The peptide databaseincludes information such as what ions comprise a particular peptide.The techniques described herein may be used to annotate the database tofurther identify which of the ions of those listed in the database areused in connection with protein profiling, for example, to characterizeor identify the protein. For example, there may be a peptide databasewhich includes a protein and identifies the 20 tryptic peptides in theprotein's sequence. It may be that only a portion, such as for example10, of those 20 peptides are ionized and may be used as precursors toidentify the protein. Using the techniques described herein, the peptidedatabase may be annotated to denote the three most intense precursors ofthe 10. The the most intense precursors may be used to identify theprotein as in connection with profiling as described elsewhere herein.The peptide database may be further annotated to identify the productions for each of the precursors as also identified, for example, usingthe retention time matching technique with the Venn intersectionprocessing as described herein.

In connection with an input data set in which a single spectrum has morethan one precursor with a same retention time, a reference injection maybe determined. Such a spectrum may be produced, for example, inconnection with an analyzed complex sample. The mass of each precursormay be determined from the reference injection. As an example, let afirst mass, m1, be associated with a first precursor and a second mass,m2, be associated with a second precursor. Multiple target injectionsmay be examined with respect to each precursor. The target injectionsmay be searched for an ion having the mass m1 and having a retentiontime within the specified second window or threshold. Such an ion isdetermined to be the first precursor in the target injection. Aretention time for the first precursor in a target injection isdetermined and aligned with the retention time of the first precursor inthe reference injection. Alignment and other processing steps may beperformed as described herein for each of the target injections todetermine which product ions are associated or matched with the firstprecursor. The same set of target injections may also be processed withrespect to the second precursor having mass m2. In a manner similar tothat as set forth regarding the first precursor with mass m1, the targetinjections may be searched for an ion having the mass m2 and having aretention time within the second window or threshold as describedelsewhere herein. A retention time for the second precursor in a targetinjection is determined and aligned with the retention time of thesecond precursor in the reference injection. Alignment and otherprocessing steps may be performed as described herein for each of thetarget injections to determine which product ions are associated ormatched with the second precursor. As such, in each of the targetinjections, appropriate product ions occurring at substantially the sameretention time as each of the precursors may be examined and processed.

To further illustrate the use of these techniques with a spectrumincluding more than one precursor, reference will be made to FIG. 16 andFIG. 17.

In FIG. 16, shown is an example spectrum that may be produced for areference injection. In the example 1200, it may be determined thatelement 1202 and 1204 are precursors having a retention time illustratedby 1240. Elements 1202 and 1204 may represent ions determined to beprecursors in accordance with their large mass and intensity in relationto other ions 1208, 1210 and 1212. In the reference injection, it is notyet clear which product ions 1208, 1210 and 1212 may be associated withwhich precursor. A first target injection is graphically illustrated inFIG. 17. When performing processing of the first target injection forthe precursor 1202, the first injection may be searched for an ionhaving the mass of 1202 and having a retention time within the secondwindow or threshold of the retention time represented by 1240. In thisfirst target injection, a precursor having the mass of 1202 also occursat a retention time 1240. However, in this target injection, onlyproduct ions 1210 and 1212, not 1208, occur at the retention time 1240within the specified window of expected error. As such, product ion 1208may not be associated or matched with precursor 1202. When performinganalysis using the first target injection with respect to precursor1204, the first target injection is searched for an ion having the massof 1204 and having a retention time within a specified window of theretention time represented by 1240. In this instance, an ion having themass of 1204 occurs at retention time 1260. Only product ion 1208 occursat retention time 1260 within the specified window of expected error. Assuch, product ions 1210 and 1212 may not be associated or matched withprecursor 1204.

It should be noted that two precursors, as well as product ions, havingdifferent retention times may have a same measured retention time in oneinjection. However, the two precursors as well as their respectiveproduct ions will have different measured retention times outside of thewindow of error, such as the 1/10^(th) peak width in repeatedexperiments. Thus, a portion of ions which seemingly are related in asingle injection may be shown to be unrelated through repeatedexperimentation.

It should be noted that a precursor ion and its one or more relatedproduct ions having a same retention time and peak shape may be referredto as a common retention time entity (CRTE). Using the techniquesdescribed herein for retention time matching, ions which seemingly areincluded in a single CRTE in a single injection can be shown to comprisetwo or more groupings or CRTEs based on different in retention times ofsubsequent injections.

As described herein, an embodiment may optionally perform processing ofan input sample prior to processing by a mass spectrometer. Suchprocessing may complement or replace separation by liquidchromatographic separation in an embodiment. In one embodiment, thesample may be a mixture of one or more molecules, such as peptides orproteins. Prior to performing mass spectroscopy, an embodiment mayseparate various proteins in the mixture using two-dimensional gelelectrophoresis (2DE). The resulting spots may be excised and digestedto break the proteins into shorter polypeptide chains. These digests maybe analyzed via mass spectroscopy. In this particular example, thesubstance may be a mixture of one or more molecules, for example, suchas peptides or proteins. An input sample or substance which includesproteins may be digested as part of enzymatic digestion processing. Thisenzymatic digestion processing is one type of separation processing thatbreaks the proteins in the sample into shorter polypeptide chains.Subsequently, the digests may then be further separated using anotherseparation processing technique such as, for example, liquidchromatography (LC), as described above, 2D Gel separation, and thelike. It should be noted that generally any separation technique and/ordigestion technique may be used to separate the various polypeptides inaccordance with, for example, molecular weight, electrical fields andthe like. The foregoing separation may optionally be performed in anembodiment on a sample prior to undergoing mass spectroscopy andgenerated spectra or other forms of data that may be included in theinput data set for retention time matching.

It should be noted that as used herein, two measured retention times maybe characterized as substantially the same if two measured retentiontimes are within the window of expected error such as described abovefor use in matching precursors with product ions. The precursor and theproduct ions may be deemed to have the same retention time even thoughthe actual measured retention times may vary within the window ofexpected error.

It should also be noted that the techniques for retention time matchingmay be applied to samples which are processed using the fractionationtechniques described herein, such as, for example, fractionation of aprotein mixture.

The processing steps performed in an embodiment, such as illustrated inFIG. 10 and FIG. 11, may be performed as a result of code executed by acomputer processor. The code may be stored on any one of a variety ofdifferent forms of computer readable media, memory and the like.

In connection with the techniques described herein in one embodiment, amolecule in a mixture may be separated in a liquid chromatograph andelute in an unmodified form. The foregoing molecule can give rise to oneor more ions in an LC/MS system and may also be referred to as theoriginating molecule. When subjected to the electrospray or otherionization processing, as well as other optional processing as may beincluded in an embodiment, a resulting mass spectrum of the originatingmolecule may include more than one ion. Multiple ions can result from,for example, the isotopic distribution of the molecule, the differentcharge states produced by ionization, and/or fragmentation mechanismapplied to the ions, or other modifications imposed subsequent toelution from the LC. Thus, an originating molecule may produce one ormore ions. In connection with the techniques described herein, the peakshapes and retention times of ions that derive from the same originatingmolecule are identical having measurements including retention timeswhich are deemed to be the same.

An ion list as described in connection with the techniques herein mayinclude one or more rows of data. In one embodiment, each row in an ionlist contains a retention time, mass/charge, and intensity describing anion. The data about each ion in the ion list may be obtained using anyone of a variety of different techniques. For example, the data aboutone or more of the ions may be obtained using the Bateman technique inthe LE or HE mode. An ion list may also refer to a list of entitiesreferred to herein as accurate-mass retention-time entities (“AMRTs”),where each row contains a retention time, mwHPlus, intensity, and chargestate as may be obtained using LE or HE acquisition mode. AMRTs aredescribed in more detail in “Quantitative Prbteomic Analysis by AccurateMass Retention Time Pairs” by Silva, et al., Anal. Chem., Vol. 77, pages2187-2200 (2005).

A spectrum included in an input data set used in connection with thetechniques described herein for retention time matching may contain alist of ions (or AMRTs), each described by an m/z (or mwHPlus) and anintensity. An embodiment may obtain a spectrum in the input data setusing a first technique including data obtained in a single scan ascollected by the mass spectrometer. In this case, the ion list for thespectrum corresponds to mass spectral peaks as may be viewed in thespectrum, and the retention time of the spectrum is the acquisition timeof the spectral scan. Alternatively, a spectrum may be obtained byselecting a retention time and a retention time window, and collectingall ions from an ion list whose retention time falls within that windowas described, for example, in PCT Patent Application No. PCTUS2005/004180, filed on Feb. 11, 2005, titled Apparatus and Method forIdentifying Peaks in Liquid Chromatography/Mass Spectrometry Data andfor Forming Spectra and Chromatograms, by Gorenstein et al. Theretention time of the spectrum may be determined to be, for example, theretention time that lies in the middle of the window represented as +/−1/10 of a chromatographic peak width measured at FWHM.

It should be noted that spectrum included in the input data set used inconnection with the retention time matching techniques described hereinmay be filtered, for example, such as by removing ions (or AMRTS) whosemasses or intensities fall outside of a particular range.

In connection with the retention matching techniques herein, the outputspectrum may be generated in a form in accordance with one or moreoutput rules. For example, as described herein, a product ion in a firstspectrum may have a first measured mass and the same product ion in asecond spectrum of the input data set may have a second measured mass.The first and second measured masses may be deemed to be the same massif they are within a defined mass tolerance. In the output spectrum, themass for the product ion may be output in accordance with a rule suchas, for example, the mass output in the output spectrum may be theaverage of the first and second measured masses. The output spectrummay, for example, consist solely of masses obtained from either thefirst or the second spectrum. Other embodiments may use other techniquesto determine the values included in the output spectrum.

The one or more spectra included in an input data set used with theretention matching techniques may come from a variety of differentsources. As described above, a spectrum may be generated in a variety ofdifferent ways from one or more experiments. A spectrum or other form ofdata included in the input data set may also come from a database orother data store. For example, data from previous experiments may bestored in a data base. The previous experimental data from the database,alone or in combination with additional new data, may be included in aninput data set. The data included in the database or other data storemay include theoretical or simulated experimental data for use inconnection with the techniques described herein. A spectrum, forexample, acquired using data-dependent acquisition (DDA) of MS/MSspectra may be included.

In an embodiment in which the sample used to obtain an input data set isa complex mixture of proteins, ions from different proteins may overlapin retention time. For such data, the retention time matching techniquesmay be applied by selecting a most intense ion in a single injection,and forming a spectrum of all ions within a retention time window ofthat most intense ion. This most intense ion may then found in asubsequent injection of substantially the same mixture of proteins bymatching masses and retention times for the precursor in both injectionsas described herein (e.g., masses of each injection within the specifiedmass tolerance, and retention times of each injection being within thesecond threshold or window as described above).

Variations, modifications, and other implementations of what isdescribed herein will occur to those of ordinary skill in the artwithout departing from the spirit and the scope of the invention asclaimed. Accordingly, the invention is to be defined not by thepreceding illustrative description but instead by the spirit and scopeof the following claims.

What is claimed is:
 1. A method of chemical analysis, comprising:providing a complex sample comprising at least a first compound and asecond compound; fractionating the complex sample into at least a firstsample portion and a second sample portion that each include portions ofthe two compounds though in different concentration ratios; performingLC/MS on the first sample portion to observe intensities of precursorions associated with the first and second compounds in the first sampleportion; performing LC/MS on the second sample portion to observeintensities of precursor ions associated with the first and secondcompounds in the second sample portion; and associating precursor ionswith their corresponding one of the first and second compounds usingratios of the observed intensities of the precursor ions in the firstand second sample portions.
 2. The method of claim 1, whereinfractionating comprises partially separating the at least two compounds,wherein the at least two compounds exhibit a peak overlap, andoversampling the overlapping peak.
 3. The method of claim 2, whereinoversampling comprises using a fraction collection time of less than apeak width to collect fractions of the overlapped peak.
 4. The method ofclaim 2, wherein the collection time is about a full-width at halfmaximum of the peak.
 5. The method of claim 2, wherein retention timesof the at least two compounds exhibiting the overlapping peak areindistinguishable.
 6. The method of claim 1, further comprising: storingin a catalog a profile of the first compound, the profile defined by anidentity of the first compound, values associated with mass andintensity of the precursor ions corresponding to the first compound, andvalues associated with chromatographic retention-times of precursorscorresponding to the precursor ions.
 7. The method of claim 1, furthercomprising: digesting the first sample portion; and fractionating thedigested first sample portion to concentrate at least one of theprecursor ions in a fraction of the first sample portion.
 8. The methodof claim 1, wherein fractionating utilizes a technique selected from thegroup of techniques consisting of: n-dimensional gel chromatography;size-exclusion chromatography; reverse-phase chromatography; andion-exclusion chromatography.
 9. The method of claim 1, wherein at leastone of the first and second compounds is a protein.
 10. The method ofclaim 1, wherein said associating includes associating a set ofprecursor ions having substantially similar intensity ratios in both thefirst sample portion and the second sample portion with a same one ofthe first and the second compound.
 11. An apparatus for performingchemical analyses, comprising: a liquid chromatography module; amass-spectrometry module in fluid communication with the liquidchromatography module to receive an eluent from the liquidchromatography module; and a computer unit, in communication with theliquid chromatography module and the mass-spectrometry module,comprising at least one processor and at least one memory for storing aplurality of instructions, which, when executed by the at least oneprocessor, causes implementation of a method comprising the steps of:digesting a complex sample comprising a plurality of polypeptides toform precursors of the plurality of polypeptides; fractionating thedigested complex sample into at least a first sample portion and asecond sample portion that each include portions of at least a firstprecursor and a second precursor of the plurality of polypeptides;performing analysis on the first sample portion using the liquidchromatography module and the mass spectrometry module to observeintensities of product ions associated with the first and secondprecursors in the first sample portion; performing analysis on thesecond sample portion using the liquid chromatography module and themass spectrometry module to observe intensities of product ionsassociated with the first and second precursors in the second sampleportion; and associating product ions with their corresponding one ofthe first and second precursors using ratios of the observed intensitiesof the product ions in the first and second sample portions.
 12. Theapparatus of claim 11, wherein the method further comprises: storing ina polypeptide catalog a profile of a first polypeptide of the pluralityof polypeptides, the profile defined by an identity of the firstpolypeptide, values associated with mass and intensity of precursor ionscorresponding to the first polypeptide, and values associated withchromatographic retention-times of precursors corresponding to theprecursors ions.
 13. The apparatus of claim 11, wherein performinganalysis on each of the first sample portion and the second sampleportion comprises forming product ions via a collision, fragmentation orreaction process.
 14. The apparatus of claim 13, wherein the collision,fragmentation or reaction process is selected from the group ofprocesses consisting of: (i) Surface Induced Dissociation (“SID”)fragmentation; (ii) Electron Transfer Dissociation fragmentation; (iii)Electron Capture Dissociation fragmentation; (iv) Electron Collision orImpact Dissociation fragmentation; (v) Photo Induced Dissociation(“PID”) fragmentation; (vi) Laser Induced Dissociation fragmentation;(vii) infrared radiation induced dissociation; (viii) ultravioletradiation induced dissociation; (ix) nozzle-skimmer interfacefragmentation; (x) in-source fragmentation; (xi) ion-source CollisionInduced Dissociation fragmentation; (xii) thermal or temperature sourcefragmentation; (xiii) electric field induced fragmentation; (xiv)magnetic field induced fragmentation; (xv) enzyme digestion or enzymedegradation fragmentation; (xvi) ion-ion reaction fragmentation; (xvii)ion-molecule reaction fragmentation; (xviii) ion-atom reactionfragmentation; (xix) ion-metastable ion reaction fragmentation; (xx)ion-metastable molecule reaction fragmentation; (xxi) ion-metastableatom reaction fragmentation; (xxii) ion-ion reaction for reacting ionsto form adduct or product ions; (xxiii) ion-molecule reaction forreacting ions to form adduct or product ions; (xxiv) ion-atom reactionfor reacting ions to form adduct or product ions; (xxv) ion-metastableion reaction for reacting ions to form adduct or product ions; (xxvi)ion-metastable molecule reaction for reacting ions to form adduct orproduct ions; and (xxvii) ion-metastable atom reaction for reacting ionsto form adduct or product ions.
 15. The apparatus of claim 14, whereinthe first polypeptide is a protein.
 16. An apparatus for performingchemical analyses, comprising: a liquid chromatography module; amass-spectrometry module in fluid communication with the liquidchromatography module to receive an eluent from the liquidchromatography module; and a computer unit, in communication with theliquid chromatography module and the mass-spectrometry module,comprising at least one processor and at least one memory for storing aplurality of instructions, which, when executed by the at least oneprocessor, causes implementation of a method comprising the steps of:fractionating a complex sample comprising at least a first polypeptideand a second polypeptide into at least a first sample portion and asecond sample portion that each include portions of the two polypeptidesthough in different concentration ratios; digesting the first sampleportion; performing analysis using the liquid chromatography module andthe mass spectrometry module on the digested first sample portion toobserve intensities of precursor ions associated with the first andsecond polypeptides in the first sample portion; digesting the secondsample portion; performing analysis using the liquid chromatographymodule and the mass spectrometry module on the digested second sampleportion to observe intensities of precursor ions associated with thefirst and second polypeptides in the second sample portion; andassociating precursor ions with their corresponding one of the first andsecond polypeptides using ratios of the observed intensities of theprecursor ions in the first and second sample portions.
 17. Theapparatus of claim 16, wherein fractionating comprises partiallyseparating the at least two polypeptides, wherein the at least twopolypeptides exhibit a peak overlap, and oversampling the overlappingpeak.
 18. The apparatus of claim 17, wherein oversampling comprisesusing a fraction collection time of less than a peak width to collectfractions of the overlapped peak.
 19. The apparatus of claim 17, whereinthe collection time is about a full-width at half maximum of the peak.20. The apparatus method of claim 17, wherein retention times of the atleast two polypeptides exhibiting the overlapping peak areindistinguishable.
 21. The apparatus of claim 16, wherein the methodfurther comprises: storing in a polypeptide catalog a profile of thefirst polypeptide, the profile defined by an identity of the firstpolypeptide, values associated with mass and intensity of the precursorions corresponding to the first polypeptide, and values associated withchromatographic retention-times of precursors corresponding to theprecursor ions.
 22. The apparatus of claim 16, wherein the methodfurther comprises fractionating the digested first sample portion toconcentrate at least one of the precursor ions in a fraction of thefirst sample portion.
 23. The apparatus of claim 16, whereinfractionating utilizes a technique selected from the group of techniquesconsisting of: n-dimensional gel chromatography; size-exclusionchromatography; reverse-phase chromatography; and ion-exclusionchromatography.
 24. The apparatus of claim 16, wherein the firstpolypeptide is a protein.